Light-Sheet Microscopy Enables Three-Dimensional Fluorescence Imaging and Live Imaging of Satellite Cells on Skeletal Muscle Fibers.

The ex vivo myofiber culture system has proven to be a useful methodology to explore the biology and behavior of satellite cells within their niche environment. However, a limitation of this system is that myofibers and their associated satellite cells are commonly examined using conventional fluorescence microscopy, which renders a three-dimensional system into two-dimensional imaging, leading to the loss of precious information or misleading interpretation of observations. Here, we report on the use of light-sheet fluorescence microscopy to generate three-dimensional and live imaging of satellite cells on myofibers. Light-sheet microscopy offers high imaging speed and good spatial resolution with minimal photo-bleaching, allowing live imaging and three-dimensional acquisition of skeletal muscle fiber specimen. The potentials of this technology are wide, ranging from the visualization of satellite cell behavior such as cell division and cell migration to imaging the sub-cellular localization of proteins or organelles.

FatJ acts via the Hippo mediator Yap1 to restrict the size of neural progenitor cell pools.

The size, composition and functioning of the spinal cord is likely to depend on appropriate numbers of progenitor and differentiated cells of a particular class, but little is known about how cell numbers are controlled in specific cell cohorts along the dorsoventral axis of the neural tube. Here, we show that FatJ cadherin, identified in a large-scale RNA interference (RNAi) screen of cadherin genes expressed in the neural tube, is localised to progenitors in intermediate regions of the neural tube. Loss of function of FatJ promotes an increase in dp4-vp1 progenitors and a concomitant increase in differentiated Lim1(+)/Lim2(+) neurons. Our studies reveal that FatJ mediates its action via the Hippo pathway mediator Yap1: loss of downstream Hippo components can rescue the defect caused by loss of FatJ. Together, our data demonstrate that RNAi screens are feasible in the chick embryonic neural tube, and show that FatJ acts through the Hippo pathway to regulate cell numbers in specific subsets of neural progenitor pools and their differentiated progeny.

A direct comparison of strategies for combinatorial RNA interference.

BACKGROUND: Combinatorial RNA interference (co-RNAi) is a valuable tool for highly effective gene suppression of single and multiple-genes targets, and can be used to prevent the escape of mutation-prone transcripts. There are currently three main approaches used to achieve co-RNAi in animal cells; multiple promoter/shRNA cassettes, long hairpin RNAs (lhRNA) and miRNA-embedded shRNAs, however, the relative effectiveness of each is not known. The current study directly compares the ability of each co-RNAi method to deliver pre-validated siRNA molecules to the same gene targets. RESULTS: Double-shRNA expression vectors were generated for each co-RNAi platform and their ability to suppress both single and double-gene reporter targets were compared. The most reliable and effective gene silencing was achieved from the multiple promoter/shRNA approach, as this method induced additive suppression of single-gene targets and equally effective knockdown of double-gene targets. Although both lhRNA and microRNA-embedded strategies provided efficient gene knockdown, suppression levels were inconsistent and activity varied greatly for different siRNAs tested. Furthermore, it appeared that not only the position of siRNAs within these multi-shRNA constructs impacted upon silencing activity, but also local properties of each individual molecule. In addition, it was also found that the insertion of up to five promoter/shRNA cassettes into a single construct did not negatively affect the efficacy of each individual shRNA. CONCLUSIONS: By directly comparing the ability of shRNAs delivered from different co-RNA platforms to initiate knockdown of the same gene targets, we found that multiple U6/shRNA cassettes offered the most reliable and predictable suppression of both single and multiple-gene targets. These results highlight some important strengths and pitfalls of the currently used methods for multiple shRNA delivery, and provide valuable insights for the design and application of reliable co-RNAi.

Cyclic Nrarp mRNA expression is regulated by the somitic oscillator but Nrarp protein levels do not oscillate.

Somites are formed progressively from the presomitic mesoderm (PSM) in a highly regulated process according to a strict periodicity driven by an oscillatory mechanism. The Notch and Wnt pathways are key components in the regulation of this somitic oscillator and data from Xenopus and zebrafish embryos indicate that the Notch-downstream target Nrarp participates in the regulation of both activities. We have analyzed Nrarp/nrarp-a expression in the PSM of chick, mouse and zebrafish embryos, and we show that it cycles in synchrony with other Notch regulated cyclic genes. In the mouse its transcription is both Wnt- and Notch-dependent, whereas in the chick and fish embryo it is simply Notch-dependent. Despite oscillating mRNA levels, Nrarp protein does not oscillate in the PSM. Finally, neither gain nor loss of Nrarp function interferes with the normal expression of Notch-related cyclic genes.

The expression of Fat-1 cadherin during chick limb development.

Cellular adhesion is fundamental to the behaviour of cell populations during embryonic development and serves to establish correct tissue pattern and architecture. The cadherin superfamily of cell adhesion proteins regulates cellular organization and additionally influences intracellular signalling cascades. Here we present for the first time a detailed account of chick Fat-1 gene expression during embryogenesis visualised by whole-mount in situ hybridisation. In part, we focus on the expression pattern in limb buds that has not been accurately documented. While Fat-1 is generally expressed in epithelial tissues and its Drosophila counterpart Fat-like regulates formation of ectodermally-derived organs, in limb buds we have found that chick Fat-1 is uniquely restricted to mesenchyme. This Fat-1 expression pattern is remarkably dynamic throughout tissue differentiation, limb maturation and pattern formation. Diffuse expression of Fat-1 begins at stage HH17 as the limb bud is forming. It then becomes more proximal as the limb bud grows and is expressed within both tendon and muscle progenitors in the dorsal and ventral subectodermal mesenchyme. Later, Fat-1 transcripts were more abundant in anterior and posterior domains of the limb bud. During hand plate formation, Fat-1 transcripts were expressed in the mesenchyme adjacent to the wrist joint zone and in the interdigit mesenchyme.

A robust system for RNA interference in the chicken using a modified microRNA operon.

RNA interference (RNAi) provides an effective method to silence gene expression and investigate gene function. However, RNAi tools for the chicken embryo have largely been adapted from vectors designed for mammalian cells. Here we present plasmid and retroviral RNAi vectors specifically designed for optimal gene silencing in chicken cells. The vectors use a chicken U6 promoter to express RNAs modelled on microRNA30, which are embedded within chicken microRNA operon sequences to ensure optimal Drosha and Dicer processing of transcripts. The chicken U6 promoter works significantly better than promoters of mammalian origin and in combination with a microRNA operon expression cassette (MOEC), achieves up to 90% silencing of target genes. By using a MOEC, we show that it is also possible to simultaneously silence two genes with a single vector. The vectors express either RFP or GFP markers, allowing simple in vivo tracking of vector delivery. Using these plasmids, we demonstrate effective silencing of Pax3, Pax6, Nkx2.1, Nkx2.2, Notch1 and Shh in discrete regions of the chicken embryonic nervous system. The efficiency and ease of use of this RNAi system paves the way for large-scale genetic screens in the chicken embryo.

Expression of avian C-terminal binding proteins (Ctbp1 and Ctbp2) during embryonic development.

C-terminal binding proteins (CtBPs) are transcriptional corepressors of mediators of Notch, Wnt, and other signalling pathways. Thus, they are potential players in the control of several developmentally important processes, including segmentation, somitogenesis, and neural tube and limb patterning. We have cloned the avian orthologues of Ctbp1 and Ctbp2 and examined their expression pattern by whole-mount in situ hybridization between Hamburger and Hamilton (HH) stages 3 and 24. Both Ctbp genes show similar expression patterns during embryonic development, and both are detected from HH stage 3 in the developing central nervous system, by HH stage 7 in the paraxial mesoderm and later in the limb bud. In most places, Ctbp1 and Ctbp2 are expressed in overlapping domains. However, there are interesting domains and/or temporal expression patterns that are specific to each Ctbp gene. For instance, Ctbp1 is predominantly expressed in the epiblast, whereas Ctbp2 is in the primitive streak at HH stage 3. However, by HH stage 4, both genes are found in the primitive streak and in the ectoderm. Similarly, although both genes display similar expression patterns in early somitogenesis, in mature somites, Ctbp1 transcripts are located in myotomal cells, whereas Ctbp2 transcripts are observed in dermomyotomal cells. Finally, we found that emigrating neural crest cells express Ctbp2, whereas dorsal root ganglia express Ctbp1. These data suggest that Ctbp1 and Ctbp2 may be functionally redundant in some tissues and have unique functions in other tissues.

Expression of avian Groucho-related genes (Grgs) during embryonic development.

Groucho-related genes (Grgs) encode transcriptional co-repressors of Lef/Tcf and Hes proteins, which are mediators of Wnt and Notch signalling, respectively. Thus, they are important players in the developmental processes controlled by Wnt and Notch signalling, including lateral inhibition, segmentation and dorso-ventral patterning. We have cloned the avian homologues of Grg genes and examined their expression pattern by whole-mount in situ hybridisation between Hamburger-Hamilton (HH) stages 3 and 24. At HH stage 3, Grg gene expression is detected in the primitive streak and Hensen's node. Later, Grg genes are expressed at high levels in the developing head fold and by HH stage 11, throughout the anterior CNS and in the ventricular zone of the neural tube. In addition, Grg2, Grg4 and Grg5 are expressed in the notochord. In the paraxial mesoderm, Grg genes are activated as soon as somites form. As somites mature, Grg1 and Grg5/AES are expressed predominantly in the medial myotome and dermomyotome, whereas Grg2, Grg3 and Grg4 are expressed throughout the myotome. In HH stage 20 limbs, Grg1, Grg3 and Grg4 transcripts are more abundant in the posterior limb bud, whereas Grg2 and Grg5/AES are expressed throughout. By HH stage 24, Grg1, Grg2 and Grg3 become localized to the dorsal and ventral limb muscle masses, whereas Grg4 and Grg5/AES occupy a more central and ventro-proximal domain, respectively. Overall, our expression data are consistent with a role for Grg genes in Lef/Tcf and Wnt signalling during somitogenesis and with a role in Hes and Notch signalling in neurogenesis.

Candidate testis-determining gene, Maestro (Mro), encodes a novel HEAT repeat protein.

Mammalian sex determination depends on the presence or absence of SRY transcripts in the embryonic gonad. Expression of SRY initiates a pathway of gene expression resulting in testis development. Here, we describe a novel gene potentially functioning in this pathway using a cDNA microarray screen for genes exhibiting sexually dimorphic expression during murine gonad development. Maestro (Mro) transcripts are first detected in the developing male gonad before overt testis differentiation. By 12.5 days postcoitus (dpc), Mro transcription is restricted to the developing testis cords and its expression is not germ cell-dependent. No expression is observed in female gonads between 10.5 and 14.5 dpc. Maestro encodes a protein containing HEAT-like repeats that localizes to the nucleolus in cell transfection assays. Maestro maps to a region of mouse chromosome 18 containing a genetic modifier of XX sex reversal. We discuss the possible function of Maestro in light of these data.